Placental mammal diversification and the Cretaceous–Tertiary boundary Mark S. Springer*†, William J. Murphy‡, Eduardo Eizirik‡, and Stephen J. O’Brien‡ *Department of Biology, University of California, Riverside, CA 92521; and ‡Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD 21702 Edited by Morris Goodman, Wayne State University, School of Medicine, Detroit, MI, and approved December 4, 2002 (received for review July 16, 2002) Competing hypotheses for the timing of the placental mammal Model by placing some intraordinal divergences (e.g., rodents, radiation focus on whether extant placental orders originated and eulipotyphlan insectivores) in excess of 100 mya (9, 11, 12). diversified before or after the Cretaceous-Tertiary (K͞T) boundary. Previous molecular studies that have addressed the timing of Molecular studies that have addressed this issue suffer from single the placental mammal radiation (reviewed in ref. 16) have calibration points, unwarranted assumptions about the molecular shortcomings that include the use of single calibration points, an clock, and͞or taxon sampling that lacks representatives of all placen- unwarranted molecular clock assumption even when there is tal orders. We investigated this problem using the largest available lineage-specific rate variation, limited taxon sampling that fails molecular data set for placental mammals, which includes segments to include all orders of placental mammals, either because of 19 nuclear and three mitochondrial genes for representatives of all molecular sequences were unavailable or because violations of extant placental orders. We used the Thorne͞Kishino method, which the molecular clock limited comparisons to reduced sets of taxa permits simultaneous constraints from the fossil record and allows [e.g., linearized tree results of Murphy et al. (14)], and failure to rates of molecular evolution to vary on different branches of a address the proposed negative correlation between body size and phylogenetic tree. Analyses that used different sets of fossil con- the rate of molecular evolution, which is potentially important straints, different priors for the base of Placentalia, and different data because mean body size increased in placental mammals subse- partitions all support interordinal divergences in the Cretaceous quent to the K͞T boundary (17). Here, we address these issues followed by intraordinal diversification mostly after the K͞T bound- using the Murphy et al. (14) data set in conjunction with recently ary. Four placental orders show intraordinal diversification that pre- developed Bayesian approaches for estimating posterior proba- dates the K͞T boundary, but only by an average of 10 million years. bilities of divergence times (18, 19). Taxon sampling includes In contrast to some molecular studies that date the rat–mouse split as representatives from each of the 18 modern placental orders old as 46 million years, our results show improved agreement with (20), as well as taxa within most of these orders that diverged the fossil record and place this split at 16–23 million years. To test the early after ordinal definition. Furthermore, the Bayesian ap- hypothesis that molecular estimates of Cretaceous divergence times proach (18, 19) avoids the molecular clock assumption and are an artifact of increased body size subsequent to the K͞T bound- permits the incorporation of multiple constraints from the fossil ary, we also performed analyses with a ‘‘K͞T body size’’ taxon set. In record. Our results, including an analysis that addressed the these analyses, interordinal splits remained in the Cretaceous. potential problem of increasing body size subsequent to the K͞T boundary and analyses that used different data partitions, are Mammalia ͉ molecular clock ͉ Placentalia ͉ phylogeny generally compatible with the Long Fuse Model and support interordinal diversification in the Cretaceous followed by in- traordinal diversification that is mostly after the K͞T boundary. rchibald and Deutschman (1) suggested the Explosive, Long AFuse, and Short Fuse models as competing hypotheses for Materials and Methods placental mammal diversification (Fig. 1). Differences between Gene and Taxon Sampling. The Murphy et al. (14) data set totals these models focus on the timing of the placental mammal radiation 16,397 aligned nucleotide positions and indexes segments of 19 relative to the Cretaceous–Tertiary (K͞T) boundary, which marked nuclear and three mitochondrial genes for 42 placental mammals. the abrupt extinction of dinosaurs. Discriminating among these Taxon sampling comprises representatives of all extant placental models is critical for understanding the historical context of pla- orders, including taxa that index the presumed deepest splits in cental mammal diversification and for deriving accurate estimates Afrosoricida (Tenrecidae ϩ Chrysochloridae), Carnivora, Cetar- of genomic and morphological rates of evolution. tiodactyla, Chiroptera, Eulipotyphla, Lagomorpha, Perissodactyla, The Explosive Model (Fig. 1a) postulates ordinal originations ͞ Primates, Rodentia, and Xenarthra. We performed analyses with and intraordinal diversification mostly or entirely after the K T the complete data set and with the following partitions: nuclear boundary. Some paleontologists favor this model (2–5, §), in genes (14,750 bp); exons (12,988 bp); first and second codon large part because essentially all fossils with characters specific ͞ positions (8,658 bp); third codon positions (4,330 bp); UTRs (1,762 to modern placental orders occur after the K T boundary. The bp); and mitochondrial RNA genes (1,647 bp). For the complete Long Fuse Model (Fig. 1b) agrees with the Explosive Model in ͞ data set, see Data Set 1, which is published as supporting informa- placing most intraordinal diversifications after the K T bound- tion on the PNAS web site, www.pnas.org. ary. However, in this scenario, interordinal divergences are placed well back in the Cretaceous. Archibald (6), Nessov et al. Branch Length Estimation. Branch lengths were estimated with the (7), and Archibald et al. (8) have interpreted 85- to 90-million- ESTBRANCHES program of Thorne et al. (18) for the complete data year-old fossils from Uzbekistan as crown-group placentals that set and each partition thereof. In each case, we used the maximum provide support for the Long Fuse Model. Finally, the Short likelihood topology from Murphy et al. (ref. 14; supporting infor- Fuse Model (Fig. 1c) postulates interordinal and some intra- ordinal divergences in excess of 100 million years ago (mya), ‘‘before or shortly after the appearance of eutherians in the fossil This paper was submitted directly (Track II) to the PNAS office. record’’ (1). Although molecular studies provide almost unani- Abbreviations: K͞T, Cretaceous–Tertiary; mya, million years ago. mous support for Cretaceous interordinal divergences (9–15), a †To whom correspondence should be addressed. E-mail: [email protected]. finding that is compatible with both the Long Fuse and Short §Novacek, M. J., Rougier, G. W., Dashzeveg, D. & McKenna, M. C. (2000) J. Vert. Paleontol. Fuse models, some molecular studies agree with the Short Fuse 3, 61A (abstr.). 1056–1061 ͉ PNAS ͉ February 4, 2003 ͉ vol. 100 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0334222100 Downloaded by guest on September 26, 2021 also performed an analysis with the diprotodontian to examine the sensitivity of our results to outgroup choice. To examine the impact of topological variation, we performed an analysis using the un- rooted Murphy et al. (14) tree for placental mammals, which was alternatively rooted at the base of myomorph rodents. This root renders Rodentia, Glires, Euarchontoglires, and Boreoeutheria paraphyletic. Although the maximum likelihood results of Murphy et al. (14) provide 100% bootstrap support for each of these clades, other studies based on both mitochondrial and nuclear DNA sequences have suggested rooting at or near the base of myomorph rodents (ref. 15 and figure 1B of ref. 24). Divergence Time Estimation. Divergence times were estimated using the program DIVTIME5B (18, 19). Markov Chain Monte Carlo analyses were run for 1 million generations after a burnin of 100,000 generations to allow Markov chains to approach stationarity before states are sampled; chains were sampled every 100 generations. Application of the DIVTIME5B program requires an input value for the mean of the prior distribution of the root of the ingroup tree. We used 105 mya, which is the mean of two extremes that were selected from the literature. First, Penny et al. (12) obtained a value of 146 mya for the placental root based on an analysis of mtDNA amino acid sequences. Second, 65 mya is based on a strict inter- pretation of the Explosive Model. To ascertain the sensitivity of our results to this prior, we also performed supplementary analyses with mean priors of 146 and 65 mya. Analyses with the ‘‘K͞T body size’’ taxon set were performed with mean priors of 65, 105, and 146 mya for the base of Placentalia. We used the following fossil constraints on divergence times (node numbers are for cross-referencing with Fig. 2): 1. Minimum of 60 mya for armadillo to sloth͞anteater (node 80) based on the occurrence of cingulatan fossils from the late Paleocene (25, 26). 2. Minimum of 50 mya and maximum of 63 mya for the split between feliform and caniform carnivores (node 59) (25, 26). Viverravids from the early Paleocene are usually regarded as stem-group carnivores, but McKenna and Bell (26) suggest